Further studies of detergent-induced conformational transitions in proteins. Circular dichroism of ovalbumin, bacterial alpha-amylase, papain, and beta-lactoglobulin at various pH values.

The conformational transitions of ovalbumin, bacterial α-amylase, papain, and β-lactoglobulin were studied in the absence and presence of sodium dodecyl sulfate (SDS) between pH 2.75 and 12.0 by means of circular dichroism (CD) measurement. The weight ratios of SDS to protein in solutions were 14:1 in all experiments. The CD bands in the near-ultraviolet spectral region were strongly reduced by SDS, whereas those in the far-ultraviolet were enhanced. With the exception of the amylase, the mean residue ellipticities of the proteins at 222 nm were increased by SDS, especially in acidic solutions. At a pH of about 3.0, the [θ]₂₂₂ values approached ?17 (±2) · 10³ deg · cm² · dmol^?1. It is assumed that at a sufficiently low pH value the proteins which are complexed with SDS have a similar backbone conformation of moderate helical content. In alkaline solutions, the detergent effect was largely reduced due to electrostatic repulsion between the negatively charged protein and dodecyl ions. The near-ultraviolet spectra of ovalbumin, papain, and β-lactoglobulin at pH 6.4 were analyzed. Assignment of the resolved bands to the appropriate chromophores was also attempted.     

Free-flow electrophoresis of proteins in phenol-containing solvents at various pH values

1. Several proteins were found to migrate when subjected to free-flow electrophoresis in buffered phenol-ethanediol-water (3:2:3, w/v/v) solvent mixtures. Mobility of these proteins changed with changing pH (apparent) values of this medium. A pH value of zero mobility for each individual protein could be estimated. 2. Founded on these observations, a high-voltage electrophoresis method in free-flowing buffer films was worked out. The method as presented here was particularly suitable for the separation of proteins on a preparative scale. Application of this and other protein fractionation techniques in dissociating media for the investigation of structural and other insoluble proteins was discussed.     

Circular dichroism of cattle rhodopsin and bathorhodopsin at liquid nitrogen temperatures

The photoevent in vision has been considered to be the conversion of rhodopsin to bathorhodopsin, which is caused by photoisomerization of the chromophoric retinal. Recently some objections were raised to this hypothesis. The reliability of the hypothesis was verified by measurement of circular dichroism of bathorhodopsin.The measurement of circular dichroism of rhodopsin extract (containing 66% or 75% of glycerol) at liquid nitrogen temperatures (?195°C) by a conventional spectropolarimeter induced an extraordinary large signal, owing to linear dichroism originated from conversion of rhodopsin to bathorhodopsin by the measuring light. The similar linear dichroism can be induced by irradiation of rhodopsin extract at ?195°C with polarized light or natural light. At photosteady state the linear dichroism disappeared.Circular dichroism spectrum of cattle rhodopsin displayed two positive peaks ([θ]_max = 80 800 degrees at 335 nm, and [θ]_max = 42 600 degrees at 500 nm) at ?195°C, whereas, bathorhodopsin displayed a positive peak ([θ]_max = 43 100 degrees at 334 nm) and a negative peak ([θ]_max = 163 000 degrees at 540 nm).The change of the positive sign to negative one at α-band of circular dichroism spectrum supports the hypothesis that the conversion of rhodopsin is due to rotation of the chromophoric retinal about C-11—12 double bond (‘photoisomerization model’).     

Circular dichroism spectroscopy of fluorescent proteins

Circular dichroism (CD) spectra have been obtained from several variants of green fluorescent protein: blue fluorescent protein (BFP), enhanced cyan fluorescent protein (CFP), enhanced green fluorescent protein (GFP), enhanced yellow fluorescent protein (YFP), all from Aequorea victoria, and the red fluorescent protein from the coral species Discosoma (DsRed). We demonstrate that CD spectra in the spectral fingerprint region of the chromophore yield spectra that after normalization are not coincident with the normalized absorbance spectra of GFP, YFP and DsRed. On the other hand, the CD spectra of BFP and CFP coincide with the absorbance spectra. The resolution of absorption and CD spectra into Gaussian bands confirmed the location of the different electronic band positions of GFP and YFP as reported in the literature using other techniques. In the case of BFP and CFP the location of Gaussian bands provided information of the vibrational progression of the electronic absorption bands. The CD spectrum of DsRed is anomalous in the sense that the major CD band has a clear excitonic character. Far-UV CD spectra of GFP confirmed the presence of the high beta-sheet content of the polypeptide chain in the three-dimensional structure.     

Circular dichroism and magnetic circular dichroism of bismuth-induced,metallothionein-like proteins

Optical studies have been carried out on bismuth-containing proteins which were isolated from the livers and kidneys of rats following injections of BiCl₃. Absorption, circular dichroism and magnetic circular dichroism spectra of hepatic Bi,Zn-metallothionein 1 and 2 indicate that the spectra are dominated by transitions from the zinc thiolate chromophore. The data from the renal Bi,Cu-metallothionein 2 are quite different and it is suggested that these spectra involve a mixture of transitions from the bismuth and copper thiolate binding sites.     

Mushroom strains able to grow at high temperatures and low pH values

Seven strains of edible mushrooms were studied with regard to mycelial growth on different growth media and culture conditions. Medium WDA (wheat/dextrose/agar) promoted higher rates of mycelial growth for all the mushrooms investigated. The majority of the strains presented higher growth rates at 30°C, but only Lentinus edodes kept maximum rates at low pH (pH 4.0), followed by Stropharia rugosoannulata and Pleurotus ostreatus (pH 5.0). Absence of light favoured rapid mycelium development in all the strains tested.     

Inhibitory effects of phenolic ecotoxicants on photobacteria at various pH values

Kinetic characteristics of light emission by intact cells of the photobacteria Photobacterium phosphoreum and Vibrio harveyi at pH 5.5, 7.0, and 8.0 were studied as well as specific features of inhibitory effects of 2,4-di- and 2,4,5-triphenoxyacetic acids (2,4-D and 2,4,5-T), pentachlorophenol (PCP), and 2,6-dimethylphenol (2,6-DMP) at the same pH values. Nonstationarity of emission kinetics was observed at all the pH values studied. Exponential luminescence decay in a 60-sec range was observed at pH 5.5; a 5-min luminescence activation, at pH 7.0 and 8.0. The cell respiratory activity drops by over one order of magnitude at pH 5.5 compared with the activities at pH 7.0 and 8.0. The inhibitory effects of 2,4-D, 2,4,5-T, and PCP differ by one-two orders of magnitude depending on pH. The maximal cell sensitivity to these compounds appears at pH 5.5; the minimal, at pH 8.0. The effect of 2,6-DMP is independent of pH. As is demonstrated, it is hydrophobicity of the molecule and pK values of the toxicants that determine the inhibitory effect. Characteristic of the substrate-starved photobacterial cells are higher sensitivity to chlorophenolic compounds compared with the cells provided with high energy supply at all the pH values.     

Kinetics of hyaluronan hydrolysis in acidic solution at various pH values

Hyaluronic acid (HA) was hydrolyzed using varying temperatures (40, 60, and 80 degrees C) and acid concentrations (0.0010, 0.010, 0.10, 0.50, 1.0, and 2.0 M HCl). The degradation process was monitored by determination of weight average molecular weight ( M w) by size-exclusion chromatography with online multiangle laser light scattering, refractive index, and intrinsic viscosity detectors (SEC-MALLS-RI-visc) on samples taken out continuously during the hydrolysis. SEC-MALLS-RI-visc showed that the degradation gave narrow molecular weight distributions with polydispersity indexes ( M w/ M n) of 1.3-1.7. Kinetic plots of 1/ M w versus time gave linear plots showing that acid hydrolysis of HA is a random process and that it follows a first order kinetics. For hydrolysis in HCl at 60 and 80 degrees C, it was shown that the kinetic rate constant ( k h) for the degradation depended linearly on the acid concentration. Further, the dependence of temperature on the hydrolysis in 0.1 M HCl was found to give a linear Arrhenius plot (ln k h vs 1/ T), with an activation energy ( E a) of 137 kJ/mol and Arrhenius constant ( A) of 7.86 x 10 (15) h (-1). (1)H NMR spectroscopy was used to characterize the product of extensive hydrolysis (48 h at 60 degrees C in 0.1 M HCl). No indication of de- N-acetylation of the N-acetyl glucosamine (GlcNAc) units or other byproducts were seen. Additionally, a low molecular weight HA was hydrolyzed in 0.1 M DCl for 4 h at 80 degrees C. It was shown that it was primarily the beta-(1-->4)-linkage between GlcNAc and glucuronic acid (GlcA) that was cleaved during hydrolysis at pH < p K a,GlcA. The dependence of the hydrolysis rate constant was further studied as a function of pH between -0.3 and 5. The degradation was found to be random (linear kinetic plots) over the entire pH range studied. Further, the kinetic rate constant was found to depend linearly on pH in the region -0.3 to 3. Above this pH (around the p K a of HA), the kinetic constant decreased more slowly, probably due to either a change in polymer conformation or due to an increased affinity for protons due to the polymer becoming charged as the GlcA units dissociated.     

Circular dichroism of beta turns in peptides and proteins.

Circular dichroism (CD) spectra are reported for two groups of cyclic hexapeptides having beta turns whose geometry can be firmly established by X-ray crystallography and by NMR spectroscopy. One series contains the sequence L-Pro-D-Phe in the geometry of the classical type II beta turn, while the second group has the sequence D-Phe-L-Pro in the closely related geometry of the gramicidin S turn. CD data on the hydrogenated peptides show that in neither series do Cotton effects due to the aromatic phenylalanyl chromophore make a significant contribution to the spectra in the 195--240-nm region. In spite of the close geometric similarity of the beta turns of these two groups of peptides, their CD spectra are quite distinct. Furthermore, comparison of our data with the CD spectra of published models for beta-turn structures suggests that it may not be possible to characterize the contribution of all beta turns to the CD spectra of proteins by a single model curve. the CD spectra of model beta turns will be more useful in characterizing the folding of oligopeptides and sequence polypeptides, where a single type of turn is present.     

Interactions of proteins with other polyelectrolytes in a two-phase system containing phenol and aqueous buffers at various pH values

1. Interactions of proteins with neutral polysaccharides and such polyacids as polygalacturonic acid, chondroitin sulphate, RNA and DNA in a two-phase system composed of phenol and aqueous buffers in the pH range 1·5–10 were studied. 2. Analysis of the products of the interaction was facilitated by the absolute preference of the proteins studied for the phenol-rich phase at all pH values. 3. The polyacids, on the other hand, in the absence of interactions were recovered mainly from the aqueous phases. 4. The interaction, the extent of which was mainly determined by the pH-dependent ionization state of the reacting partners, followed the patterns of antigen–antibody interactions with a well-defined equivalence point (maximum point of precipitation) and with the formation of soluble complexes. 5. The soluble complexes formed below the equivalence point were composed of proteins with small amounts of polyacids attached, and so passed into the phenol-rich phase; those formed above the maximum precipitation point were polyacidic in character and found in the aqueous phases. 6. Glycoproteins, with small amounts of covalently linked sugar residues, passed quantitatively into the phenol-rich phases. 7. The possibilities of developing a method for the analysis of glycoproteins and other applications are discussed.     

Dodecyl sulphate/polyacrylamide-gel electrophoresis at low pH values and low temperatures.

A simple method is described for dodecyl sulphate/polyacrylamide-gel electrophoresis of pH- and temperature-labile biological intermediates. The method is based on a catalyst system that works at temperatures of 2--4 degrees C and pH values of 2--4 and an appropriate buffer system containing Li+ or Tris [CH2OH--C(CH2OH)2--NH3+] instead of Na+. This system does not lead to the precipitation of 1% dodecyl sulphate.     

Long-time molecular dynamics simulations of botulinum biotoxin type-A at different pH values and temperatures

Botulinum neurotoxins type A (BoNT/A) are highly potent toxins, but are also useful in the treatment of illnesses. We studied the properties of BoNT/A at various temperatures and pH values in order to understand its toxicity and structure variations. The pH values of the environment of BoNT/A are obtained by changing the protonation states of certain titratable residue groups. Our results show that certain parts of the protein are active at acidic pH environments or at high temperatures. The protein is more stable in neutral environments at normal human body temperature, whereas, at high temperature, the protein is more stable in acidic environments. Also, the three domains of the protein tend to have relative motion rather than within individual domains.